Because antibodies specific for a large variety of biological substances can be prepared, polyclonal antibodies produced in rabbits, goats, or sheep have been very important reagents in all disciplines of biomedical research and in medical diagnostics. These antibodies are the essential components in various kinds of immunoassays, such as radioimmunoassays and enzyme-linked immunosorbent assays.
In the past 10-15 years, the development of monoclonal antibody methodologies has further enhanced the importance of antibody reagents in biomedical research medical diagnostics. These monoclonal antibodies (MAbs) have additional utilities in in vivo diagnostics and therapies.
In producing MAbs in mice, the first step is to inoculate the mice with the immunogen which one wishes to make monoclonal antibodies against. Thereafter, following a well-known procedure, one fuses B cells from the mouse spleen with a tumorous B cell line (myeloma cell line) to create immortal hybridoma cell lines, and then screens and identifies the hybridomas which produce the monoclonal antibodies of interest. The screening of these hybridomas is typically done by determining the specific reactivity of the antibodies secreted by the hybridomas with the immunogen, or a peptide representing the epitope of the immunogen which is of interest.
Many immunogens are not capable of triggering an adequate antibody response in the mice. This means that there are only few B cells producing antibody against the immunogen, making it difficult to isolate these cell lines after forming hybridomas. The low antibody response results because the immunogens do not elicit adequate T cell help to expand B cell clones specific for the antigen to an appreciable extent.
Thus, one often uses adjuvants which cause an enhanced antibody response against an immunogen, in order to increase the number of B cells which produce antibody against the immunogen. One potent adjuvant which is often used for priming a response is Freund's complete adjuvant ("CFA"). CFA is a mixture of oil (Bayol F) and detergent (mannide monooleate) containing Mycobacterium tuberculosis. CFA is administered in a mixture together with the immunogen. There is a need to investigate new adjuvants, however, because regulatory agencies discourage use of CFA in laboratory animals, due to its serious side effects.
This application is directed to adjuvants which are conjugates of polymers and binding molecules which target surface antigens on T cells, preferably CD3 or other components of the T cell antigen receptor. Binding molecules are antibodies, antibody fragments, single chain antibodies, and peptides which are capable of receptor binding. Such adjuvants have never been disclosed or suggested, as discussed below.
A number of MAbs specific for CD3 on the surface of human T cells (pan T markers) are known to be very potent mitogens of human T cells in vitro, e.g., the MAb OKT3, Van Wauwe, J. P. et al., J. Immunology 124:2708 (1980); Chang, T. W. et al., Proc. Natl. Acad. Sci. U.S.A. 78:1805 (1981); MAb 64.1 Hansen, J. A. et al., Leukocyte Typing: Human Leukocyte Differentiation Antigens Detected by Monoclonal Antibodies, Eds. Bernard, A. et al. (Spring Verlag, N.Y., 1984). In medium containing only fetal calf serum and no human serum (and therefore no IgG), the anti-CD3 MAbs are much more potent than phytohemagglutinin A ("PHA") or Concanavalin A ("Con A") in inducing T cell proliferation.
But the mitogenic effect of anti-CD3 requires both specific binding to the CD3 antigen and the presence of the Fc moiety of the antibody, as well as the presence of monocytes and macrophages. The best explanation for these results is that the Fc of the anti-CD3 MAbs binds to the Fc receptors on monocytes/macrophages, thereby aggregating the CD3 antigen and the T cell receptor (CD3/TCR) on the T cell surface, which triggers the activation and proliferation of the T cells.
This explanation is supported by experiments which show that when the anti-human CD3 MAb is conjugated to Sepharose 4B beads or coated on the substratum plastic surface of culture wells, monocytes and macrophages are not needed to induce activation and proliferation of T cells. See Williams, J. M. et al., J. Immunol. 135:2249 (1985); Ceuppens, J. L. & Baroja, M. L., J. Immunol. 137:1816 (1986); Geppert, T. D. & Lipsky P. E., J. Immunol. 138:1660 (1987). Based on these experiments, it has been suggested that the solid-phase anti-CD3 MAb functions by aggregating the CD3/TCR complexes on the T cell surface.
However, when anti-human CD3 is injected in vivo, the results are the opposite of the in vitro effects. OKT3 MAb, which is the first MAb ever approved for therapeutic use in vivo, is strongly immunosuppressive and is approved for use as an immunosuppressant for patients receiving kidney transplants. Ortho Multicenter Group Study, N. Eng. J. Med. 313:337 (1985). The injection of OKT3 causes rapid depletion of T cells from the circulation.
Administration of anti-CD3 monoclonal antibodies (or F(ab').sub.2 fragments thereof) to mice also causes T cell depletion. Hirsch, R. et al., Transplantation 49:1117-23 (1990). This monoclonal antibody also caused weight loss, diarrhea, and decreased activity within 24 hours in all mice, and death of one-half of the mice it was administered to in 3 days. Hirsch, R. et al., id. at p. 1118, col. 1, paragraph 2. In another study it was observed that mice administered 40 .mu.g of this anti-CD3 antibody seemed to develop more rapidly growing tumors. Ellenhorn, J. D. et al., Science 242:569-571 (1988) at p. 570, in the legend of FIG. 3. These observations are consistent with the observation that this antibody caused depletion of T cells and immunosuppression.
Although the mechanism by which anti-CD3 causes this rapid depletion of T cells is not well understood, the best explanation is that anti-CD3 induces antibody-dependent cellular cytotoxicity ("ADCC") of the T cells, i.e., as the T cells coated by anti-CD3 circulate through the spleen and liver, they are lysed by the phagocytic cells of the reticuloendothelial system in these organs. It is also possible that some of the T cells are destroyed by complement-mediated cytolysis and some other cytolytic mechanisms.
Thus, the studies all indicate that anti-CD3 binding molecules will cause depletion of T cells in vivo. Depletion of T cells causes a generalized immunosuppression, and does not lead to an increase in the number of antibody-producing B cells. Accordingly, one would expect that anti-CD3 binding molecules would not act as effective adjuvants for aiding in producing antibodies.